We plan to study the structure and the regulation of the rat prolactin gene, using rat pituitary tumor cells. Poly(A ion) RNA enriched in prolactin mRNA translational activity will be extracted from the cells. DNA complementary to it will be prepared and fragments of the cDNA, originating from prolactin mRNA will be purified. These fragments will be used as markers for the subsequent isolation and cloning of double-stranded DNA coding for the prolactin mRNA, and also for the isolation and cloning of the prolactin gene isolated from rat DNA. The nucleic acid sequences of the prolactin mRNA and the prolactin gene will be compared. Regulation of prolactin gene expression will be studied using 1) immunoassay for measurement of prolactin production by the cells; 2) cell-free translation and immunoprecipitation of preprolactin for measuring the translational activity of the prolactin mRNA; and 3) molecular hybridizations with the pure cDNA probe obtained from the cloned material, to measure the number of nucleic acid sequences. The questions asked will involve: kinetics of induction and deinduction of cytoplasmic mRNA; regulation and processing of the primary prolactin nuclear transcript; expression and regulation of the prolactin gene in isolated nuclei and eventually in native and reconstituted chromatin; and number and nature of the prolactin gene in chromatin.